Therapeutic and nutritional compositions

ABSTRACT

Novel nutritional compositions and methods for their manufacture, administration and use are disclosed. The nutritional compositions of the present invention are useful for the feeding of immature mammals, particularly those used for food, and/or performance animals, such as horses, and for companion animals, such as dogs and cats. The nutritional compositions and supplements of the present invention are effective in supporting the growth and health of the immature mammal, in supporting and stimulating its immune system, and in mitigating undesirable infections and other. Additionally, particular fractions of the unique peptides produced by the disclosed methods can be separated and utilized as therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/658,093, filed Apr. 16, 2018, the disclosure of which is herebyincorporated by reference in its entirety, including all figures, tablesand amino acid or nucleic acid sequences.

FIELD OF THE INVENTION

The present invention relates to nutritional compositions for enhancinghealth and development in domestic animals. The nutritional products andsupplements are prepared by unique milk fermentation processes.

BACKGROUND OF THE INVENTION

Fermentation processes are used throughout the world for the manufactureof foods based on milk from a number of different mammalian sources,including domestic as well as wild animals. These processes typicallyinvolve adding lactic acid-producing microorganisms, such as bacteriaand yeast to milk, which ingest lactose, or milk sugar, and releaselactic acid as waste. Depending on fermentation conditions and milksource, different products are obtained.

Traditional uses of fermented mare's milk are believed to havebeneficial effects in relief of metabolic and intestinal problems,gut-cleansing effects coupled with repair of intestinal flora, relieffrom stomach ulcers, and normalization of blood pressure, cholesteroland liver problems. It has been recommended as an aid in cancertreatment, likely due to enhanced nutrition and support of the immunesystem.

More recently, researchers have utilized fermented equine milk for thetreatment of certain human pathologies, such as hepatitis, chronic ulcerand tuberculosis (Nassal and Rembalski, 1980; Solaroli, Pagliarini andPeri, 1993).

Fermentation agents are typically lactic acid bacteria, such asLactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris,Lactococcus lactis biovar diacetylactis, and L. acidophilus, and yeasts,such as Leuconostoc mesenteroides, subsp. cremoris. Mixtures of bacteriaand/or yeast are used for many different products, includingLactobacillus bulgaricus and Streptococcus thermophilus for yoghurt, anda mixture of bacteria and yeasts for kefir and kumis.

Since ancient times, agrarian cultures have utilized milk for nutrition.Due to a lack of preservation methods and modern methods, such asrefrigeration, yeast fermentation was typically used to provide aproduct that could be safely used over a period of time. However, thefermented milk products differed in quality and characteristicsdepending on the milk source. Production methods were not controlled anddepended on naturally occurring yeasts and bacteria already present inthe milk.

Even today in the human populations of central Asia and the formerSoviet Union, fermented horse milk is mainly used for the manufacture ofa lactic-alcoholic beverage containing from 2-5% alcohol, known as Airagor Koumiss. Typically, the fermentation bacteria utilized are lacticacid bacteria, such as Bifidobacterium mongoliense sp., Lactobaccilusheveticus and Lactobacillus kefiranofaciens. Surono and Hosono (2003)disclose a combination of Lactobacillus lactis subsp. Lactis,Lactobacillus delbrueckii subsp. bulgaricus and the yeast Torula spp.for the production of Koumiss.

Commercially produced fermented products for human consumption includecheese, sour cream, yoghurt and similar products in countries throughoutthe world, which are known as dahi (Pakistan), kefir (Russia), and crémefraiche (France). Differences between these food products arise from theuse of milk with different milk fat contents and the fermentation agent.In many cases the bacteria and/or yeast are unknown or are alreadypresent in varying degrees in the collected milk. Some products areconsistent in quality after controlled manufacture, e.g., sour cream,but have a short shelf life due to continuing action of the bacteriaand/or yeast, e.g., créme fraiche, which has a typical shelf life of 10days at 4° C. while sour cream has a shelf life of 4 weeks. Cheeseexhibits a wide variation depending on milk source and milk fat content,which can range for 1 to 75% milk fat.

In addition to human food, animals have been fed different fermentedfeeds, traditionally produced by yeasts naturally present in grass cropsthat have been stored under moist conditions. Dairy farms havesupplemented animal diets with rumen-specific live yeast, typicallySaccharomyces cerevisiae (CNCM 1-1077), to improve subacute rumenacidosis in cows.

Interest has focused recently on animal feed efficiency and the value offermented animal fodder for improving animal health and development.Fermented liquid feed for pigs was shown to increase weight 3-5% byusing lacto-fermented feedstock (J. Animal Science and Biotechnology,2015, 6-4). Fermented liquid feed can be produced from agriculturalfoodstock mixed with water, lactic acid bacteria and yeasts. The pH ofliquid feed can be reduced so that stomach pH more efficiently inhibitsproliferation of pathogens in the animal.

Nutritional benefits of milk alone are well recognized. Althoughmammalian milk is comparable in different species, equine milk naturallyhas better nutritional value than bovine milk.

Horse milk is superior to bovine milk in several characteristics,including: (1) more thermostable whey proteins; (2) less sensitivity tothermal processing; (3) lower casein content; (4) characterization as analbuminous milk; (5) ability to transmit in-utero chemosensoryinformation relative to future food and “additive” products; (6)contains cathelicidins that work like natural antibiotics; (7) equalamounts of β-casein and α-casein; (8) contains N-acetylneuraminic acid(sialic acid) with a high concentration of 0-acetylation at position 4;(9) higher concentrations of lactoferrin and lysozyme; and (10) lysozymeis about 10% compared to trace amounts of lysozyme in bovine milk.

Tables 1-4 illustrate some of the differences in composition betweenhorse and bovine milk.

Table 1 shows the differences between mare's milk and bovine milk.

TABLE 1 Parameter Equine Milk Bovine Milk Crude protein (g/kg) 21.4 32.5True whey protein (g/kg) 8.3 5.7 Casein (g/kg) 10.7 25.1 NPN × 6.38(g/kg) 2.4 1.7 (non-protein nitrogen) True whey protein (%) 38.79 17.54Casein (%) 50.00 77.23 NPN × 6.38 (%) 11.21 5.23 Total solids (g/kg)110.0 Lactose (g/kg) 63.7 48.8 Fat (g/kg) 12.1 36.1 Ash (g/kg) 4.2 7.6Gross energy (kcal/kg) 480.0 674.0

Table 2 compares the lipid concentration of mare's milk and bovine milk.

TABLE 2 Parameter Equine milk Bovine milk Fat (g/kg) 12.1 36.1Triglycerides (%) 81.1 97.0 Phospholipids (%) 5.0 1.5 Unsaponifiable (%)4.5 1.5 Free Fatty Acids (%) 9.4 Trace Palmitic FA (%) 23.8 29.5 OleicFA (%) 19.1 26.3 Linoleic FA (%) 9.6 2.9 Linolenic FA (%) 9.4 1.1Saturated FA (%) 55.8 68.0 Unsaturated FA (%) 44.2 32.0

Table 3 compares the vitamin and mineral content of mare's milk andbovine milk.

TABLE 3 Parameter Equine milk Bovine milk Calcium (mg/L)  500-1,3001,100 Magnesium (mg/L)  40-110 100 Zinc (mg/L) 0.9-6.4 4 Vitamin E(mg/L) 0.26-1.13 0.6 Riboflavin (mg/L) 0.37 1.83

Table 4 shows the differences in casein species and micelle size betweenmare's milk and bovine milk.

TABLE 4 Parameter Equine milk Bovine milk Casein (g/kg) 10.70 25.10α_(s) - casein (%) 46.65 48.46 β - casein (%) 45.64 35.77 κ- casein (%)(7.71) 12.69 Micelles size (nm) 255.00 182.00

BRIEF SUMMARY OF THE INVENTION

The nutritional compositions of the present invention are effective insupporting the growth and health of immature mammals, particularly inimproving and stimulating the immune system, and mitigating undesirableinfections and other diseases that are commonly prevalent in the typicalenvironment of the immature mammal. The formulations are designed forefficient administration to herds, as well as to individual animals.Certain components found in the novel nutritional compositions arise inpart from the processing techniques which separate less desirable milkcomponents and concentrate bioactive peptides which contribute to thenutritional and health benefits.

Particular fractions of the described fermented milk products can beseparated and utilized as therapeutic agents; for example, foranti-anxiety activity in animals. This is particularly important incompanion animals which commonly exhibit separation anxiety andnoise-phobic behavior, in addition to benefiting modulation of theimmune system and growth promotion.

The described compositions are typically prepared from mammalian milkand/or colostrum, which is fermented using a unique mixture of bacterialfermentation strains. Colostrum and/or milk components can be separated,and certain fractions can be utilized in new combinations to formnutritional compositions. These nutritional compositions comprisebioactive peptides which arise from changes the milk proteins haveundergone during fermentation and processing.

The compositions of the present invention are formulated to enhancegrowth and strengthen the immune system in immature animals,particularly equine foals and other immature herd animals. Accordingly,the compositions are particularly useful as either a complement to, orsubstitute for key components of mammalian milk. The novel compositionspromote cell maturation and support nutrient uptake during growthperiods, as well as aiding in gut maturation. This ensures not only thehealth and growth of a neonate, but also the development of the activelygrowing young animal, maintenance of the adult, and enhancement ofsurvivability and quality of life of mature or senior animals. Thecompositions promote uptake and increase the bioavailability ofnutrients and production of required micronutrients.

An important aspect of the nutritional products of the present inventionis the strengthening of the immune system which obviates commoninfections normally treated by antibiotics. The compositions will alsoassist in the protection of the intestinal mucosa from aggressiveactions of potentially dangerous substances and pathogens and activateprotective macrophages to fight infections and increase the immunesystem. They may scavenge free radicals that could damage cell tissueand reduce cell immunity. By protecting intestinal tissue, thecompositions rebuild tissue damaged from chronic diarrhea, and preventthe return of the diarrhea condition and its resulting damage tointestinal tissue.

An advantage of the compositions of the present invention is theconvenient manufacture under commercial conditions in large quantities.This makes sufficient quantities available for treatment of performanceanimals, such as horses and production animals, such as cattle, goats,and sheep, including companion animals in need of such nutritionalsupport. The compositions of the present invention can be formulated forconvenient administration to both individual animals and herds ofanimals.

Additional features of the nutritional formulations of the presentinvention include stability under a range of usage conditions and acommercially acceptable shelf life. Certain formulations can bedeveloped that also do not require refrigeration by the user throughoutthe shelf life prior to usage.

The described invention utilizes isolated milk peptides and bioactivecomponents in the formulations from fermented mare's milk and bovinecolostrum. These compositions provide a natural defense againstIrritable Bowel Syndrome (IBS) and Irritable Bowel Disease (IBD), andrepair damaged tissue from the use of NSAIDs (aspirin, etc.) and otherconditions that cause intestinal damage. The bioactive ingredients inthe disclosed novel compositions are extremely important as a preventiveof squamous and glandular ulcers in horses. Certain isolated peptidesand bioactive products arising from the fermentation process can beformulated to have physiological effects, such as behavior modification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions produced from mammalianmilk or colostrum, using a process which includes fermentation underdefined conditions with one or more unique strains of bacteria. Theproduct can be used directly as a feed or supplement or fractionatedinto different components which contribute more significantly tonutritional value compared to unfermented milk or colostrum.

The novel compositions are particularly useful in enhancing growth andstrengthening the immune system in immature mammals, such as neonatalfoals, calves, lambs, kids, puppies, kittens, camelids, and otheranimals that depend on colostrum for survival and growth. Through theadministration of these compositions to juvenile mammals, nutritionaluptake, and hence growth and maturation, is optimized, as well asstrengthening of the immune system, optimization of anabolicbody-building pathways, and protection against a number of conditionsaffecting the health of the animal.

The described nutritional compositions comprise milk peptides andbioactive components separated from fermented mare's milk and bovinecolostrum. These compositions provide a natural defense againstIrritable Bowel Syndrome (IBS) and Irritable Bowel Disease (IBD). Use ofthe compositions repairs damaged tissue arising from use of NSAIDs(aspirin, etc.) and other conditions that cause intestinal damage. Thisis extremely important in preventing squamous and glandular ulcers inhorses. Behavior modification has also been observed when thecompositions are used to treat stress and anxiety in domestic andcompanion animals.

The compositions contain concentrated mixtures of novel peptidesproduced by the new multi-stage fermentation method, in addition tonaturally-occurring milk proteins in untreated milk, but lack thedeleterious components commonly found in fermented milk products.

Enhanced nutritional compositions can be obtained from fermentation ofanimal feedstock, such as fodder, grass, or grain by-products which aretreated with a unique mixture of Przewalski bacteria, particularly oneor more or mixtures of the strains Ruminococcus flavefaciens (ATCCAccession No. 49949), Butyrivibrio fibrisolvens (ATCC Accession No.19171), and Fibrobacter succinogenes (ATCC Accession No. 51216). Aliquid culture mixture of the bacteria can be applied to feedstock,which is then used directly as feed or as a feed additive.

An unexpected effect of one component produced by the describedfermentation method is its anti-anxiety effect. A significant calmingeffect is observed when administered to animals in certain high-stresssituations which can cause acute anxiety, e.g., anxiety, fear, orabnormal behavior having a sudden onset, such as a visit to aveterinarian or groomer, separation from family, kenneling, shows, loudnoises, such as fireworks or thunderstorms, pet adoptions, new homes,new dog houses, carriers, or crate cages, exposure to vehicle rides,relocation, etc. The fractionated product can also be utilized as apreventive agent for animals that are undergoing shipment or othertransfer where they could injure themselves as a result of beingfrightened and/or anxious.

Products are typically produced from pasteurized and defatted mammalianmilk, preferably from mare's milk or colostrum because equine milk'snatural nutritional value is superior to that of bovine milk. The milkor colostrum is optionally fermented with lactobacillus bacteria beforecombining Przewalski bacterial cultures and subjected to heat andenzymatic processing.

The Przewalski bacteria used in the process for making the describednutritional products were originally isolated and cultured from thefeces of the Przewalski horse, Equus ferus przewalskii, which isconsidered to be the last truly wild horse in existence and even todaysurvives only in limited zoo environments. This rare and endangeredequine, the ancestor of today's domestic horse, originally existed onlarge numbers in the steppes along the Mongolia/China border. Today theanimal is officially considered extinct because the last individualsighted was over 50 years ago.

Mammalian milk or colostrum, after fermentation with a Przewalskiculture, or optionally with lactic acid bacteria, preferablyLactobacillus delbrueckii subsp. bulgaricus, and Streptococcusthermophilus, is subjected to enzymatic proteolysis in a series ofincubation steps followed by separation procedures described herein, toafford various semi-purified fractions which concentrate bioactivecomponents. Further fractionation yields unique nutritional compositionswhich have desirable nutritional effects. The nutritional and biologicaleffects of the fractions isolated from the described fermentationprocess are different from the effects of unfermented milk fractions.

The components of the compositions of the present invention providenutritional supplementation for immature mammals and all mammals in needof the unique bioactive peptides critical for optimized health, growthand performance. These compositions enhance efficient growth, providebioactive components that enhance, support and stimulate a neonate'sgrowth and optimized differentiation of muscle groups, and/or strengthenand modulate the immune system, while providing basic and limitingnutrients to the neonate, rapidly maturing individual or animalsrequiring precise targeted bioactive nutrients that are typically rareand unavailable to a normal population of the same species.

The present invention also makes compromising environmental conditionsless likely to negatively affect animals that consume the bioactiveingredients on a regular basis. The invention evens out the effect of aninconsistent or ever changing environment by providing the naturallyrare concentrations of glyconutrients, bioactive peptides,immunoglobulins, sialic acid with the o-acetylation at position 4,cytokines, proline rich polypeptides, thymosin subunits, lactoferrin,transferrin, xanthine oxidase, lactoperoxidase, insulin-like growthfactors, glutathione precursors, beta-lactoglobulins,alpha-lactoglobulins, lysozymes, beneficial microbials and prebiotics.In addition to these there are hundreds of metabolites and peptides thatare derived from the fermentation of the mare's milk with the very rareinoculants (bacteria) which were isolated from the Przewalski horse.

The nutritional compositions of the present invention may alsooptionally include one or more ingredients to enhance the nutrition ofthe immature mammal, e.g., the equine foal, bovine calf or ovine lamb.Such ingredients may include vitamins and/or mineral supplements. Forexample, vitamin E can be added to the supplement, as can variousminerals, such as selenium, copper, manganese, zinc, and/or chromium. Incertain instances, it may also be desirable to include enzymesupplements to aid in digestion.

Other ingredient(s) of the nutritional product may include vitamins andminerals which contribute to the final nutritional product in theircapacity as antioxidants and/or enhance the total nutritional qualitiesof the nutritional product. One of the additional ingredients used in apreferred embodiment is Vitamin E. Other ingredients may be used in thepreferred embodiment, including various minerals, such as selenium,copper, manganese, zinc, and chromium. Especially for equines it may bedesirable to include mannan oligosaccharides, typically those derivedfrom the cell wall of the yeast Saccharomyces cerevisiae, and varioussupplemental dietary enzymes which are known to assist in aidingdigestion of the grain-based diets commonly fed to mammals, especiallyhorses. Typical enzyme supplements are those supplying bacterialBacillus licheniformis alpha-amylase, alone or in combination withamyloglucosidase (AMG, another starch-digesting enzyme found in thesmall intestine), which are known to improve starch digestion in thehorse.

Echium plantagineum seed oil (echium oil) is a terrestrial plant sourcethat may hold promise as a sustainable alternative to fish oil becauseit has a very high content of unsaturated fatty acids (92% of totalfatty acids; 50% of which are ω-3 FA), and is rich in stearidonic acid(15-20%) and gamma-linolenic acid. Stearidonic acid (SDA, C18:4 n3), aprecursor of the bioactive lipids eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) which are found in fatty cold-water fish.These acids are recognized as having possible functions to improvemetabolism and delay the onset or prevent diabetes. Research in mice hasfound that echium oil improved glucose handling in the body. This issignificant insofar as there are currently 84 million Americans who haveprediabetes (1 in 3).

Use of echium oil and the bioactive peptide blends will improve glucosehomeostasis, suggesting that combinations may (1) enhance systemicglucose uptake and possibly B-cell function, (2) lead to reducedvisceral adipose accumulation, and (3) increase tissue PUFA accumulationwhile reducing tissue saturated fatty acid (SFA) accumulation.

Combinations of camel milk and echium oil are proposed as an alternativeintervention for diabetes in humans and animals. The novel peptidesproduced by fermentation of bovine colostrum as described herein arealternative substitutes to camel milk.

Echium oil and the novel peptides produced by fermentation of bovinecolostrum are showing promise as a novel and safe intervention in horseswith Equine Metabolic syndrome (EMS), insulin dysregulation, anddiabetes mellitus. By changing the concentration and ratios ofingredients it is possible to intervene in horses that have problemswith EME related laminitis. EMS and pituitary pars intermediadysfunction (also known as equine Cushing's disease), which also causesregional adiposity, laminitis, and sometimes insulin resistance, can bemanaged successfully with combinations of selected peptides and echiumoil.

The combination of the ingredients discussed above in the preparation ofthe nutritional composition of the present invention for immaturemammals will enhance both the growth and immunity of the animals, andresult in healthy, productive and performance oriented adult animals,with a more defined easily obtainable quality of life when allowed tohave optimized environmental conditions. The nutritional compositioncomplements and compensates for deficiencies in the mother's milk,neonatal diets, and challenging environmental conditions, while alsoproviding the rare bioactive substances that aid in protection frompotentially dangerous pathogens in the feedstuffs available in themother's and neonate's feed or forage.

The nutritional product of the present invention can be administered inany of a number of ways, including adding to feed, or fed directly as anutritional composition. It is desirable that the immature mammal isgiven regular doses of the nutritional composition, typically daily ortwice daily, for periods of 1-60, preferably 1-30, days after birth.

The nutritional composition of the present invention may be manufacturedas a liquid or paste and stored in a gelatin capsule (as a gelcap),which provides a consistent dosage of the nutritional product.Alternately, it can be manufactured as a paste for oral administrationusing a dose syringe. Finally, it may be manufactured pelleted togetherwith grass meal and/or alfalfa meal. The pelleting procedure should beperformed at low temperature, preferably not higher than 65° C. Theingredients mentioned above should be approximately 5-60 percent, and,most preferably, about 20 percent, by weight of the pellet formulation.

The present invention teaches a nutritional product which enhancesgrowth, while modulating and strengthening the immune system in neonatesand animals that will directly benefit from the invention's rare andunique compilation of nature's most profoundly influential bioactivesubstances. As such, the nutritional compositions of the presentinvention are usable as either a complement to or substitute formother's milk and a neonatal first diet other than mother's milk. Itpromotes proper cell maturation and metabolism while supporting nutrientabsorption and assimilation during growth periods, homeostasischallenges, performance activities, as well as aiding in gut adaptation,maturation, and differentiation, and ensuring the health, growth, andquality of life of the immature, growing and mature mammal. It helpscarry and increase the bioavailability of nutrients and help producerequired micronutrients and further to sequence essential unidentifiedrare bioactive ingredients that have never been seen before in nature,but are created by nature and science coming together in the currentinvention.

More specifically, the nutritional compositions of the present inventionalso strengthen and modulate the immune system, and protect theintestinal mucosa from the aggressive actions of potentially dangeroussubstances and pathogens. These compositions activate protectivemacrophages to fight infections and boost the immune system, andscavenge free radicals that can damage cell tissue and reduce cellimmunity. The nutritional compositions of the present invention alsoprotect intestinal tissue, rebuild damaged tissue after chronicdiarrhea, and prevent the diarrhea condition and the resulting damage tointestinal tissue. The oligosaccharide components of the compositionsmimic lectin binding sites and prevent many pathogenic organisms frombinding and causing damage.

The herein described nutritional products efficaciously treat andprevent digestive tract ulcers in foals and other immature animals. Theinvention is also effective in treating inflammatory bowel disease. Theproducts consist entirely of safe and naturally derivatized ingredientsrather than chemically manufactured drugs. The nutritional products ofthe present invention are administrable in a variety of dosage forms,thereby making their dispensation to both individual animals and herds asimple matter.

The nutritional product of the present invention is stable and has along shelf life, and requires no special care to be provided by the userthroughout its shelf life prior to usage.

The relative ranges of amounts of each of the components of thecompositions, and their preferred amounts are discussed, beginning withthe bioactive peptide component. The range of amounts of bioactivepeptides is between approximately one-half percent and ten percent ofthe nutritional composition by weight. The preferred amount of thebioactive peptide component is approximately five percent of thenutritional product by weight.

The range of β-lactoglobulin is between approximately 30 and 70%,preferably about 40-60% of the nutritional product by weight. The mostpreferred amount of β-lactoglobulin is approximately 50-53% of thenutritional composition by weight.

The range of α-lactalbumin is between approximately 10 and 40%,preferably about 20-30%, of the nutritional composition by weight. Themost preferred amount of α-lactalbumin is approximately 22-25% of thenutritional product by weight.

The preferred range of the amount of immunoglobulins is betweenapproximately 10 and 30%, and more preferably about 14-25%, of thenutritional composition by weight. The most preferred amount ofimmunoglobulins is approximately 16-20% of the nutritional product byweight.

The preferred range of serum albumin is between approximately 2 and 10%,preferably about 3-8%, of the nutritional composition by weight. Themost preferred amount of serum albumin is approximately 4-6% of thenutritional composition by weight.

The range of amounts of lactoferrin is between approximately 5 and 15percent of the nutritional composition by weight. A preferred amount oflactoferrin is approximately 10 percent of the nutritional compositionby weight.

The range of amounts of lysozyme is between approximately 10 and 11percent of the nutritional composition by weight.

The range of amounts of Vitamin E is between approximately one-twentiethpercent and one-half percent of the nutritional composition by weight.The preferred amount of Vitamin E is approximately two-tenths of apercent of the nutritional composition by weight.

The nutritional product of the present invention may be administeredeither by adding it to feed or by feeding it directly as a nutritionalproduct. In a preferred embodiment, the nutritional product isadministered once or twice daily. It may be manufactured either as aliquid, in which case it can be added to feed which is then fed to ahorse, or as a liquid or paste and stored in gelatin capsules (asgelcaps), which makes for a consistent and uniform dosage of thenutritional product. If manufactured as a paste, it can also be orallyadministered using a dose syringe.

Alternatively, a nutritional product of the present invention may bemanufactured by pelleting it together with grass meal and/or alfalfameal. The pelleting procedure should be performed at a low temperature,preferably not higher than 65° C., to avoid the degradation ordestruction of the beneficial ingredients, particularly those containedin the whey protein concentrate. The ingredients of the nutritionalproduct should be approximately twenty percent by weight of the totalweight of the pellets.

The typical dosage of the nutritional product of the present inventionis approximately ten grams per day for foals from birth to three monthsold, approximately twenty grams per day for foals from three months tosix months old, and approximately forty grams per day for foals from sixmonths to a year old.

It will be readily apparent to those skilled in the art from thepreceding discussion of the ingredients of the nutritional product ofthe present invention and their interaction that the benefits achievedby the nutritional product of the present invention is substantiallygreater than the sum of the benefits of each of the nutritionalproduct's ingredients separately.

The description of the preferred embodiments of the present inventiondisclose nutritional products which both enhance growth and strengthenthe immune system in equine foals and, potentially, in other animals andeven humans as well. As such, the products of the present invention areusable as either a complement or substitute for mare's milk; promotecell maturation and support nutrient uptake during growth periods, aswell as aiding in gut maturation and ensuring the health and growth offoals or young horses. The products help carry and increase thebioavailability of nutrients and helps produce required micronutrients.

The disclosed products strengthen the immune system and help protect theintestinal mucosa from the aggressive actions of potentially dangeroussubstances and pathogens; activate protective macrophages to fightinfections and boost the immune system, and scavenge free radicals thatcould damage cell tissue and reduce cell immunity. The products alsoprotect intestinal tissue, rebuild damaged tissue after chronicdiarrhea, and prevent the diarrhea condition and the resulting damage tointestinal tissue.

The nutritional product of the present invention efficaciously treatsand prevents digestive tract ulcers in foals and, potentially, in otheranimals and even humans. It consists entirely of safe and naturalingredients rather than drugs. The product is orally administrable,thereby making its dispensation a simple matter.

The nutritional products of the present invention are stable and have along shelf life, and require no special care by the user throughoutshelf life prior to usage. The products are also inexpensive relative toconventionally used nutritional products for foals, thereby enhancingmarket appeal and affording the broadest possible market. Finally, allthe aforesaid advantages and objectives of the nutritional productsmethod of administration are achieved without incurring any substantialrelative disadvantage.

Materials

Mannan oligosaccharides (MOS) are derived from the cell wall of theyeast Saccharomyces cerevisiae (UK Vet Chem, Mulund West, Mumbai,Maharashtra, India).

INTIzyme enzymes (Integrated Laboratories PVT, Ltd, Shakti, Nagar,Ambala, Haryana, India) contain fungal diastase, pepsin and B complex.INTIzyme enzymes are a commercially available mixture of maltase,lactase, α-glucosidase, β-glucosidase, lipase, cellulose, protease andglycerophosphorase which facilitates the conversion of nutrientmaterials to sugars that are used for energy, amino acids which providebody building materials, and other materials, such as phosphoric acid,which provides regulatory functions within the animal, such as themaintenance of stable blood pH.

PRP is a proline-rich polypeptide mixture containing growth factorsIGF-1 and 1111 IgG (DNA containing 6 tandem repeats affectingdevelopment). A PRP mixture is separated as a fraction from thefermented milk processed as described herein and contains about 25%ulin.

Przewalski culture is a culture medium or milk aerobically fermentedwith one or more of Ruminococcus flavefaciens (ATCC Accession No.49949), Butyrivibrio fibrisolvens (ATCC Accession No. 19171),Fibrobacter succinogenes (ATCC Accession No. 51216) and Ruminococcusalbus (ATCC Accession No. 27211).

“Bioactive Component 1” is a mixture of amino acids and peptidesfractionated from milk or colostrum after fermentation with Przewalskibacterial cultures and processing with hydrolytic and proteolyticenzymes.

Lactobacillus delbrueckii, subsp. bulgaris (ATCC Accession No. 11842)and Streptococcus thermophilus (ATCC Accession No. 19528) are lacticacid bacteria used to ferment milk or colostrum.

Ingredient 1111 can be obtained from colostrum whey and providesb-lactoglobulin, a=lacctalbumin, albumin and immunoglobulins.

Echium oil is obtained from echium plantagineum plant seed and isavailable commercially (Jedwards International, Inc., Braintree, Mass.).

EXAMPLES

The present invention utilizes compositions that are prepared frommammalian colostrum and/or mammalian milk, using a fermentation processwith Przewalski bacteria. The examples describe the compositions,processes for making the fermented products and beneficial effects ofusing the enhanced nutritional products.

Example 1 Przewalski Bacterial Cultures

The “Przewalski culture” used in the present invention is a mixture ofone or more of bacterial strains which have been isolated fromPrzewalski horses. Fecal samples from Przewalski horses are inoculatedonto agar and incubated in an anaerobic chamber at about 37° C. forabout 40-60 hours. The resultant sub-cultured colonies are then platedonto MRS agar (available from Sigma-Aldrich). The MRS broth is thenadjusted to about pH 2.0, 3.0, 4.0 or 5.0 by the addition ofhydrochloric acid, resulting in isolates which are then plated and grownin pure culture on MRS agar for about 50 hours at about 37° C. Thestrains are purified into single cultures by plating.

The bacteria comprising the “Przewalski culture” as employed hereincomprise a mixture of Ruminococcus flavefaciens (ATCC Accession No.49949), Butyrivibrio fibrisolvens (ATCC Accession No. 19171),Fibrobacter succinogenes (ATCC Accession No. 51216) and Ruminococcusalbus (ATCC Accession No. 27211). Each of these organisms is availablefrom the American Type Culture Collection (ATCC), or other similarculture collections throughout the world. Alternatively, one or more ofPrzewalski bacteria may be used where indicated as Przewalski culturesin the disclosed formulations.

Example 2 Fermentation Mash

Oat fiber is sprayed with the Przewalski culture of Example 1 grown toabout 7 to 9 log cfu in liquid culture under aerobic fermentationconditions. The resulting mixture is a mash containing about 1-3% of thebacterial strains. The mixture is dried and ground to produce a “DriedFermentation Mash” additive to supplement up to 20% of an animal feed.Fermentation mash metabolites are activated in the gut of the treatedanimal and contribute to health and immune system benefits.

Alternatively, the mash is not dried but used directly as a liquid feedor added to conventional feed as a liquid.

Example 3 Preparation of Bioactive Peptide Mixtures

Fermentation of mammalian colostrum or milk, particularly equinecolostrum or milk with the Przewalski culture results in a mixture whichhas an increased silylation rate compared to the silylation rates of themilk components prior to fermentation. Further enzymatic processing ofthe fermented milk, using a sequence of proteolytic enzymes and heatingfollowed by ultrafiltration, provides filtrates which consist ofconcentrated peptides formed during the fermentation process. Bioactivepeptides can then be separated from the other components of thefermented milk using chromatography and HPLC to obtain mixtures ofpeptides in the range of 6000 Daltons as described in Example 5.

Example 4 Preparation of Proline-Rich Polypeptide (“PRP”) Solution

1 kg mare's milk is heated at 90° C. for 3 min, then inoculated with 10g of Lactobacillus delbrueckii, subsp. bulgaris (ATCC Accession No.11842) and Streptococcus thermophilus (ATCC Accession No. 19528)cultured in skim milk for 8 hrs at 42° C. to 9.0 and 7.0 log cfu mg/ml,respectively. The mixture is stirred, bottled and heated at 42° C. to apH of 4.2. The mixture is cooled to 4-6° C. and can be stored for atleast 45 days.

The milk is treated to remove lipids. The separated lipids, comprisingfatty acids, glycolipids and phospholipids are removed from thecolostrum or milk. The defatted milk contains immunoglobulins (IgG),proline-rich polypeptides, glycoproteins, oligosaccharides, enzymes,interferons, cytokines, bactalbumins, probioticbacteria/microbiome/growth factors and certain vitamins and minerals.Chromatography of the defatted milk provides a protein rich fractionrich in proline-rich polypeptides (PRP). This fraction is furtherfractionated to provide a “PRP” solution containing 70-100% proline-richpolypeptides.

Example 5 Method for Preparing Bioactive Component 1

Pasteurized and defatted colostrum or milk is fermented with thePrzewalski culture of Example 1 and processed with rennet to precipitatecasein. The filtrate containing lactoferrin, which has a molecularweight of about 80,000, is taken up in distilled water at 5% (wt/vol),and the pH is adjusted to about 2.5. This solution is then subjected toporcine and/or cod pepsin (EC 3.4.23.1, 10 units/mg) in acidic pH,followed by aspartic protease (PD enzyme) of Penicillium duponti at 10units/mg at a temperature of about 37° C. for a period appropriate tothe specific enzyme.

The resultant acidic pH enzymatically treated composition is thenadjusted to a pH of 7.0, and incubated with protease A (10 units/mg,Amano Pharmaceutical); papain (EC 3.4.22.2, 50 units/mg, NagaseBiochemical Co.); Actinase AS (230 units/mg, Kaken Pharmaceutical Co.);trypsin (EC 3.4.21.4, 1000 units/mg, Sigma Chemical Co.) and Bioprase(20 units/mg, Nagase Biochemicals Co.). This hydrolysis reaction isconducted at a temperature of about 37° C. for a period appropriate tothe specific enzyme.

The reaction is neutralized by the addition of 1N sodium hydroxide tostop further hydrolysis and/or any other enzymatic activity. Heating toabout 80° C. for a period of about 10-60 minutes, preferably a period ofabout 15 minutes, stops further hydrolysis, leaving a compositioncontaining approximately forty distinct bioactive peptides having arange of molecular weights of less than about 6000 kD. The degree ofhydrolysis ranges from <1% to about 40% consisting of about 1% to about35% free amino acids. These distinct bioactive peptides (“BioactiveComponent 1”) can be utilized as mixtures, and included as an ingredientin the herein-described nutritional products.

The Bioactive Component 1 mixture can be fractionated by conventionaltechniques, such as column chromatography, ultrafiltration, orcombinations thereof, to produce pure or substantially pure bioactivepeptide mixtures having varying molecular weights in the approximaterange of <500 to about 10,000 daltons.

Example 6

Defatted equine colostrum can be treated with rennet, and precipitatedcasein removed by filtration, resulting in colostrum whey. The maincomponents of colostrum whey are β-lactoglobulin, α-lactalbumin, albuminand immunoglobulins. These are typically separated by isoelectric pointusing ion exchange media in a stirred tank reactor Table 5 shows therelative proportions of the proteins and their isoelectric points.

A lactose solution is added to the colostrum solution. The dilutedprotein liquor is subjected to ultrafiltration and concentrated. Furthernanofiltration/diafiltration can be used to isolate the various peptidesshown in Table 5.

TABLE 5 Component Proportion (%) Isoelectric Point β-lactoglobulin 50-535.2-5.4 α-lactalbumin 22-25 4.5-5.1 Albumin 4-6 4.9-5.1 Immunoglobulins16-20 5.8-8.3

Example 7 Equine Nutritional Composition—Formula 1040

The ingredients shown in Table 6 are combined in dry form and thoroughlymixed. The composition is mixed with conventional feed or usedseparately and administered on a daily basis.

TABLE 6 Ingredient % by weight Bioactive component I 83.302 INTIzymeenzymes 10.433 1111, IGg 0.8676 g., PRP 4.338 mg, IGF-1 0.867 μg 2.169Przewalski cultures 2.272 Mannan oligosaccharide 1.824 Total 100.000%

Five mature purebred quarter horses were fed daily with the nutritionalcomposition added to regular feed as a supplement to provide about 15 toabout 30 grams per animal, adjusted for the body weight and physicalactivity of the animal. Two horses were fed the same amount of feedwithout the supplement. Two horses did not have a measured control dietand were used as controls. The five horses receiving the supplementedfeed showed at least 5% superior growth rates and better physiologicalhealth than the two horses receiving the same diet without thesupplement and were also healthier than the two control horses.

This composition provides isolated milk peptides and bioactiveingredients that potentiate genetically predisposed capabilities forenhanced feed efficiency, improve fiber digestibility, allow phenotypicenhancement and allow growing animals to reach their genetic potentialfor muscle definition and function. The composition enhances anabolicmetabolic agents in muscles; supplies agents that help the body torepair damaged tissue from the use of NSAIDs (aspirin, etc.) and otherconditions that cause intestinal damage; prevents Irritable BowelSyndrome (IBS) and Irritable Bowel Disease (IBD); supports intestinalintegrity and helps reestablish epithelial health; optimizes support formultiple intestinal repair pathways and helps support a healthy gutbarrier border, thereby contributing to overall immune health; reducesgut inflammation; resulting in a reduction of allergic symptoms; helpsto partition aids in cell protection and increased proliferation of cellmembrane protection components; and can aid in behavioral modification.

Example 8 Equine Nutritional Composition—Formula 1014

Table 7 shows the composition of a nutritional product particularlysuited for maintenance of mature horses.

TABLE 7 Ingredient % by weight 1111, IGg 17.62 g., PRP 88.1 mg, IGF-117.62 μg 44.05 MOS 1.90 Bioactive Component I 41.85 Przewalski Cultures2.20 INTIzyme enzymes 10.00 Total 100.00

This composition, when utilized at the described dosage, providesisolated milk peptides, eight essential saccharides and bioactiveingredients that potentiate genetically predisposed capabilities forenhanced feed efficiency; improve fiber digestibility; allows phenotypicenhancement and allows growing animals to reach their genetic potentialfor muscle definition and function; enhances anabolic metabolic agentsin muscles; supplies agents that help the body to repair damaged tissuefrom the use of NSAIDs (aspirin, etc.) and other conditions that causeintestinal damage and squamous and gastric ulcers in horses; preventsIrritable Bowel Syndrome (IBS) and Irritable Bowel Disease (IBD);supports intestinal integrity and helps reestablish epithelial health;optimizes support for multiple intestinal repair pathways and helpssupport a healthy gut barrier border, thereby contributing to overallimmune health; reduces gut inflammation resulting in a reduction ofallergic symptoms; helps to partition nutrients to target tissuesessential for proper immune function; enhances immune modulation; aidsin cell protection and increased proliferation of cell membraneprotection components; and can aid in behavioral modification, resultingin a decrease of many consequences of unwanted spontaneous activity.Further, it reduces inflammation and promotes healing in the performancehorse and similar animal athletes and supports overall enteric health.

Example 9 Anxiolytic Composition

The active ingredient of the anxiolytic product is a decapeptide presentin a low concentration in the Bioactive Component 1 mixture of Example5. When the Bioactive component 1 mixture is fractionated using columnchromatography, the decapeptide fraction can be purified to a level ofabout 70-100%, typically containing about 3-10 mg decapeptide/ml ofliquid, Specific Gravity=0.99-1.11.

Optimal administration is 45 minutes to 1 hour prior to activity; for anacute situation, double the dose 1 hour before event. Stressfulsituations are unique to individual animals. Feeding levels can beincreased with no risk. Shake container before each use. In general, 2teaspoons for a 1200 lb. horse. Doses typically utilized in equines areshown in Table 8.

TABLE 8 PONIES & SMALL HORSES: 5 TO 10 mL (2 teaspoons = 10 ml) AVERAGESIZE HORSES: 10 TO 30 mL (2 tablespoons = 30 ml) LARGE HORSES: 30 TO 50mL

For animals experiencing nondynamic environmental stressors that lead todeath can be utilized to treat and/or prevent anxiety in both companionanimals, such as dogs and cats, and production animals, such as cattle,goats and sheep, as well as equines.

Anxiolytic Composition

TABLE 9 Active Ingredient Amount/100 g formulation Decapeptidehydrolysate of 8.5 g equine milk casein comprising αS1-casein,αS2-casein, β-casein, κ-casein 3-10 mg/ml solution Sialic acid 1600-1750mg IGF-1 25-38 μg Proline Rich Polypeptides 2-3.5 g (“PRP”) solution

Microbial Standards

TABLE 10 Microbial Standards Typical Method(s) APC <5000 cfu/g   AOAC986.33, 989.10 Coliforms Absent/g AOAC 991.14 Escherichia coli Absent/gAOAC 991.14 Yeast and molds <10 cfu/g  AOAC 997.02 Salmonella  Absent/25 g AOAC 989.13 Listeria   Absent/25 g AOACVIP 997.03Coagulase positive Absent/g AOAC 2003.08 Staph. aureus

Chemical Standards

TABLE 11 Chemical Standards Typical Method(s) Protein 8.5% AOAC Fat 1.4%AOAC Carbohydrates 74.1%  Calculation Ash  14% SMEDP Moisture  2% AOAC

A study was conducted using the anxiolytic composition to demonstratethe anti-anxiety effects on a group of 20 dogs. Within an hour ofadministration up to 90% of the dogs exhibited significantly reducedsigns of stress or anxiety. 27% of the dogs were observed to be calmerwithin 20 minutes of administration. 86% of the dogs were observed to becalmer within 40 minutes of administration. 90%+of the dogs wereobserved to be calmer within 50 minutes of administration.

In a similar study using cats, similar results were obtained withrespect to the anxiolytic effects, and most importantly, a very lowincidence of side effects was observed as compared to a placebo, withonly about 4.45% of the anxyliotic compound treated cats exhibitinglethargy and a similar percent exhibiting excessive water intake. Theplacebo treated animals, in comparison, had 13.63% which exhibited “offfeed/less intake”, 9.09% exhibiting constipation, 4.45% exhibiting “notusing litter box” and 9.09% exhibiting excessive water intake.

Example 10 Fermented Milk Preparation

1 kg mare's milk is heated at 90° C. for 3 min, then inoculated with 10g of Lactobacillus delbrueckii subsp. bulgaris (ATCC Accession No.11842) and Streptococcus thermophilus (ATCC Accession No. 19528)cultured in skim milk for 24 hrs at 40° to 45° C. to 9.0 and 7.0 logcfu/g, respectively. The mixture is stirred, bottled and heated at 42°C. to a pH of 4.2. The mixture is cooled to 4-6° C. and can be storedfor at least 45 days.

Although the foregoing description of the nutritional compositions andthe anti-anxiety compositions of the present invention have been shownand described with reference to particular embodiments and applicationsthereof, it has been presented for purposes of illustration anddescription and is not intended to be exhaustive or to limit theinvention to the particular embodiments and applications disclosed. Itwill be apparent to those of ordinary skill in the art that a number ofchanges, modifications, variations, or alterations to the invention asdescribed herein may be made, none of which depart from the spirit orscope of the present invention. The particular embodiments andapplications were chosen and described to provide the best illustrationof the principles of the invention and its practical application tothereby enable one of ordinary skill in the art to utilize the inventionin various embodiments and with various modifications as are suited tothe particular use contemplated. All such changes, modifications,variations, and alterations should therefore be seen as being within thescope of the present invention as determined by the appended claims wheninterpreted in accordance with the breadth to which they are fairly,legally, and equitably entitled.

What is claimed is:
 1. An anxiolytic composition for ameliorating stressor anxiety in a companion animal or a production animal, saidcomposition comprising sialic acid, insulin-like growth factor 1(IGF-1), a proline-rich polypeptide (PRP) solution comprising 70-100%proline-rich polypeptides, and a decapeptide hydrolysate of caseincomprising αS1-casein, αS2-casein, βS2-casein and κ-casein, wherein thePRP solution is obtained by fermenting defatted mammalian milk withLactobacillus delbrueckii and Streptococcus thermophilus and wherein thecomposition is in an amount sufficient to ameliorate the stress or theanxiety in said companion animal or said production animal.
 2. Theanxiolytic composition of claim 1, wherein the companion animal is adog, equine, or cat.
 3. The anxiolytic composition of claim 1, whereinthe production animal is a cow, goat or sheep.
 4. The anxiolyticcomposition of claim 1, wherein the composition upon administration tothe companion animal or the production animal in amounts of 5 to 50grams, 45 minutes to one hour prior to an acute situation, amelioratesthe stress or the anxiety.
 5. The anxiolytic composition of claim 4,wherein the acute situation is abnormal behavior having a sudden onset.6. The anxiolytic composition of claim 5, wherein the acute abnormalbehavior includes fear and reaction to loud noise.
 7. The anxiolyticcomposition of claim 2, wherein the amount sufficient to ameliorate thestress or the anxiety in an equine is 10 to 50 grams depending on itssize and age.
 8. The anxiolytic composition of claim 7, wherein theamount sufficient to ameliorate the stress or the anxiety in an averagesized 1200 pounds equine is 30 grams.
 9. The anxiolytic composition ofclaim 1, wherein the proline rich polypeptide (PRP) is 2 to 4 weightpercent, the sialic acid is 1.6 to 1.75 weight percent, the IGF-1 is 2.5to 3.8×10⁻⁴ weight percent and the decapeptide hydrolysate is 8 to 10weight percent of the composition.
 10. The anxiolytic composition ofclaim 9, wherein the decapeptide hydrolysate of casein is obtained byhydrolysis of casein precipitated from defatted equine colostrum. 11.The anxiolytic composition of claim 1, wherein the defatted mammalianmilk is mare's milk.